MS and UPLC-DAD

Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 207–215

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Chemical profiling and quantitation of bioactive compounds in Platycladi Cacumen by UPLC-Q-TOF-MS/MS and UPLC-DAD Bo Zhuang a,1 , Zhi-Ming Bi a,1 , Zi-Yuan Wang a , Li Duan b , Chang-Jiang-Sheng Lai c , E-Hu Liu a,∗ a State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing, 210009, China b College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang, 050024, Hebei, China c National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, State Key Laboratory Breeding Base of Dao-di Herbs, Beijing, 100700, China

a r t i c l e

i n f o

Article history: Received 11 January 2018 Received in revised form 28 February 2018 Accepted 2 March 2018 Available online 9 March 2018 Keywords: Platycladi cacumen Chemical profiling Quantitation Quality control

a b s t r a c t Platycladi Cacumen (PC) is a traditional Chinese medicine used for the treatment of hemorrhages, cough, asthma and hair loss. To get a better understanding of the chemical constituents in PC, ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and diagnostic ion filtering strategy were firstly employed for chemical profiling of PC. A total of 43 compounds including organic acids and derivatives, flavonoids as well as phenylpropanolds were unambiguously or reasonably identified. Coumarin and lignan were reported for the first time in PC. Chemical variation of 39 batches of PC from different geographical origins and 10 batches of processed product of PC was subsequently investigated by quantitation of nine major flavonoids. The results determined by UPLC coupled with diode array detection (UPLC-DAD) and hierarchical cluster analysis (HCA) indicated that the contents of flavonoids in PC samples differ greatly. This work provides an efficient approach to comprehensively evaluate the quality of PC. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Platycladi Cacumen (PC), derived from the dried branches and leaves of Platycladus orientalis (L.) Franco, has been ubiquitously used in China for a long time. As a traditional Chinese medicine (TCM) and food additive, PC was recorded and summarized in ancient manuscript such as “Shen Nong Ben Cao Jing” and officially listed in the Chinese Pharmacopoeia [1]. PC has been applied in treatment of cough, gout, asthma, chronic bronchitis, hemorrhage disease and hair loss [1]. Recently, there has been growing evidence in different biological activities of PC, including antiphlogistic [2–4], antioxidant [5–7], hemostatic [8], neuroprotective [9,10] and hair growth promoting [11–13]. For example, the flavonoids in PC were reported to have a significant anti-inflammatory effect on lipo-polysaccharide-induced macrophage cells [2]. Also, essential oils extracted from different parts of PC demonstrated distinct antioxidant activity [7]. Cecarbon was confirmed to be a hemostatic

∗ Corresponding author. E-mail address: [email protected] (E.-H. Liu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jpba.2018.03.005 0731-7085/© 2018 Elsevier B.V. All rights reserved.

compound by spectrum-effect relationship study [14]. Moreover, as a major constituent from PC, cedrol was considered as a hair growth promotor for its remarkable effects on hair growth and hair follicle length [11]. Platycladi Cacumen Carbonisatus (PCC), the legal processed product of PC, exhibits enhanced anti-hemorrhagic activity in pharmacological and clinical studies [14,15]. It has been well acknowledged that the efficacy of herbal medicines is significantly relevant to the chemical composition and the contents of active compounds in herbs. Compared with its long history of clinical use, chemical analysis and quality control studies on PC are rather limited. In previous literature, chemical analysis of PC was rarely performed for determination of one or a few of flavonoids, which are considered as the bioactive constituents responsible for the efficacy of PC [16–21]. There is still a lack of systematical research on the chemical profiling of PC. Also, the chemical variance of PC and PCC is seldom reported. In the present study, both qualitative and quantitative analyses of chemical constituents were conducted for comprehensive quality control of PC. Ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and diagnostic ion filtering strategy were employed for phytochemical profil-

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Table 1 Thirty-nine batches of PC. Number

Origins

Number

Origins

PC01 PC02 PC03 PC04 PC05 PC06 PC07 PC08 PC09 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC19 PC20

Jiangsu Jiangsu Zhejiang Anhui Anhui Anhui Jiangxi Shandong Shandong Shandong Shandong Shandong Shandong Shanxi Shanxi Shanxi Hebei Hebei Hebei Henan

PC21 PC22 PC23 PC24 PC25 PC26 PC27 PC28 PC29 PC30 PC31 PC32 PC33 PC34 PC35 PC36 PC37 PC38 PC39

Henan Hubei Hubei Hunan Hunan Hunan Guangxi Guangxi Guangdong Guangdong Sichuan Guizhou Guizhou Yunnan Shanxi Gansu Gansu Jilin Jilin

ing of PC. Furthermore, 9 major flavonoids containing myricitrin, isoquercitrin, quercitrin, myricetin, afzelin, quercetin, kaempferol, amentoflavone and hinokiflavone in 39 batches of PC and 10 batches of PCC, were simultaneously quantitated using an UPLC coupled with diode array detection (UPLC-DAD) method. Hierarchical cluster analysis (HCA) was carried out to distinguish different batches of PC. Data presented in this investigation give a close insight into the phytochemical characterization of constituents in PC with the emphasis on flavonoids. 2. Experimental 2.1. Materials and reagents A total of thirty nine batches of PC collected in present study were purchased from various Traditional Chinese Medicine market in eighteen province of China. The batch numbers were listed in Table 1. Ten batches of PCC were carbonized according to method of the Chinese Pharmacopoeia (2015 edition). Reference compounds of shikimic acid, citric acid, protocatechuic acid, procyanidin B2, rutin, epicatechin, p-coumaric acid, myricetrin, quercitrin, apigenin-7-O-ˇ-d-glucoside, myricetin, quercetin, kaempferol, kaempferide, and amentoflavone were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and hinokiflavone was acquired from Meilune Biotech Co. Ltd (Dalian, China). Isoquercitrin was purchased from Must Bio-technology Co. Ltd (Chengdu, China). Kaempferol-3O-rhamnoside (afzelin) was previously isolated from Ardisiae Japonica Herba extract in the authors’ laboratory. The purity of all reference compounds were determined to be >98% by high performance liquid chromatography-diode array detection analysis. Deionized water used in experiments was purified by a Milli-Q water purification system from Millipore (Bedford, MA, USA). The LC/MS-grade acetonitrile was purchased from Merck (Darmstadt, Germany). The HPLC-grade formic acid was purchased from ROE Scientific Inc. (Newark, New Castle, USA). HPLC-grade acetonitrile was purchased from TEDIA (Fairfield, OH, USA). All other reagents and chemicals used were of analytical grade.

until use. A mixed standard solution was prepared by diluting stock solutions to desired concentrations with methanol. The dry plant material was firstly ground into powder and sieved (60 mesh). For qualitative analysis, a total of 0.5 g plant material powder was accurately weighed, and then extracted by ultrasonicating (KQ5200B, 200W, 40 kHz, Kunshan, China) for 40 min with 20 mL 75% methanol at room temperature for each sample. For quantitative analysis, samples of 0.5 g of powdered PC and PCC were extracted with 10 mL of 75% ethyl alcohol by ultrasoundassisted extraction (UAE). The mixture was ultrasounded at room temperature for 60 min. All the prepared samples were followed by centrifugation at 16200 G for 10 min, and then analyzed directly. Each extraction was performed by triplicate. 2.3. Instrumentation and chromatographic conditions 2.3.1. Chemical profiling by UPLC-Q-TOF-MS/MS Chromatographic analysis was performed on an Agilent 1290 Series HPLC system equipped with a diode array detector, a quaternary solvent delivery system and a column temperature controller. All the samples were carried out at a column temperature of 35 ◦ C on a Thermo BDS Hypersil C18 column (250 mm × 4.6 mm, 5 ␮m, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of water with 0.2% formic acid (eluent A) and acetonitrile (eluent B) using a gradient elution mode of 10% B at 0–4 min, 10%–20% B at 4–24 min, 20% B at 24–30 min, 20%–23% B at 30–32 min, 23%–35% B at 32–44 min, 35%–50% B at 44–48 min, 50% B at 48–52 min, 50%–56% B at 52–55 min, 56%–95% B at 55–57 min, 95%–100% B at 57–60 min, 100% B at 60–65 min. The flow rate was 0.8 mL/min and the injection volume was 5 ␮L. An Agilent 6530 QTOF tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) was applied to MS and MS/MS detection. The operation conditions were as follows: drying gas (N2 ) flow rate, 10 L/min; drying gas temperature, 350 ◦ C; nebulizer, 35 psig; sheath gas flow rate, 11 L/min; sheath gas temperature, 350 ◦ C; capillary voltages, 4000 V; fragmentor, 135 V; skimmer, 65 V; OCT RF Vpp, 750 V. The data were acquired in negative ion mode; collision energy was 15 V, 25 V and 35 V. Mass spectra were recorded across the range of m/z 50–1500. Agilent MassHunter Workstation Acquisition Software Version B.05.01 and Qualitative Analysis Software Version B.07.00 were utilized for system control, data acquisition, and data processing. 2.3.2. Quantitative analysis of nine flavonoids constituents in PC and PCC Quantitative analysis was carried out on an Agilent 1290 Series HPLC system equipped with a diode array detector, a quaternary solvent delivery system and a column temperature controller. Chromatographic separation was conducted on a Thermo BDS Hypersil C18 column (100 mm × 4.6 mm, 2.4 ␮m, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase was composed with solvent A (0.2% aqueous formic acid) and solvent B (acetonitrile) with a gradient elution program: 0–3 min, 17% B; 3–6 min, 17–19% B; 6–7 min, 19–23% B, 7–11 min, 23–35% B; 11–13 min, 35–40% B; 13–14 min, 40% B; 14–16 min, 40–47% B; 16–19 min, 47–50% B; 19–20 min, 50–100% B; 20–24 min, 100% B. The constant flow rate was 0.8 mL/min and the column was maintained at 35 ◦ C. The injection volume was 5 ␮L and the detection wavelength was set at 254 nm at 0–14 min and 340 nm at 14–24 min. 2.4. Data analysis

2.2. Preparation of standard solutions and sample solutions Individual stock solutions of the references used for quantitative analysis were prepared by dissolving the each reference in methanol at a concentration of 1 mg/mL and then stored at 4 ◦ C

HCA was carried out based on the peak area of quantification of nine flavonoids compounds in all samples using SPSS Statistics 22 (IBM, Chicago, IL, USA) Software. When the target compounds were not detected or the contents of them less than the LOD in

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the samples, the values of such elements were considered to be 0. Total contents of nine flavones were performed by GraphPad Prism software (version 6.00, La Jolla California, USA).

current (TIC) chromatogram of PC extract in negative ion mode by UPLC-ESI-Q-TOF-MS. The compounds characterized are presented in Table 2, considering the elution order.

3. Results and discussion

3.1.2. Identification of organic acids and derivatives The dominating product ions of organic acids corresponded to the loss of CO2 (44 Da), CO (28 Da) and H2 O (18 Da), which are helpful for further identification certainty. Compound 1 was identified as quinic acid based on its [M−H]− ion at m/z 191 and the resulting product ions at m/z 173 due to the neutral loss of water moiety as well as the fragment at m/z 127 ([M−H–CO–H2 O]− ), which is consistent with the data reported in the literature [23]. Quinic acid derivatives p-coumaroylquinic acids (compound 11 and 17) were also tentatively identified due to the loss of 146 Da and the formation of a diagnostic ion at m/z 191 (deprotonated quinic acid) [24]. Compound 2 ([M−H]− ion at m/z 195) corresponded to gluconic acid, produced fragment ions at m/z 177 ([M−H−H2 O]− ) and m/z 129 ([M−H−2H2 O−CH2 O]− ) [25]. Compound 3 showed a [M−H]− ion at m/z 133 and was characterized as malic acid. The fragment ion at m/z 115 was yielded due to the loss of water. Compound 4 was assigned as shikimic acid and its MS/MS spectrum showed a fragment at m/z 137 corresponding to the loss of CO2 moiety. Citric acid (Compound 5) exhibited a [M−H]− ion at m/z 191 and produced a key fragment at m/z 111 ([M−H−CO2 −2H2 O]− ) [26]. Compound 8, with [M−H]− ion at m/z 153, produced a fragment ion at m/z 109 owing to the loss of CO2 , was unequivocally identified as protocatechuic acid by comparing with the reference standard. Compound 6 and 7, with the same [M−H]− ion at m/z 315, were reasonably characterized as protocatechuic acid-hexoside, which are protocatechuic acid derivatives with a main MS/MS pattern at m/z 153, corresponding to the loss of hexoside (162 Da) residue. Compound 19 was characterized as p-coumaric acid by comparing with MS/MS spectrum of the standard and literature [24]. While compound 12, displaying a [M−H]− at m/z 163, yielded a fragment at m/z 119 corresponding to the loss of CO2 moiety and was identified as p-coumaric acid isomer.

3.1. Chemical profiling of PC by UPLC-Q-TOF-MS/MS 3.1.1. Research strategy Although liquid chromatography coupled with mass spectrometry has been proved as a powerful tool for structural characterization of unknown compounds, the reliable identification of chemical components in herbal material still remains a great challenge due to its inherent complexity [22]. In the present study, the diagnostic ion filtering strategy was employed to analyze the untargeted mass spectral data of compounds in PC, in combination with the biosynthetic pathway of natural compounds. Firstly, the fragmentation behaviors of authentic compounds in PC were systematically investigated and the chemical compounds isolated from PC were summarized from previous reference literatures. Essential information of chemical constituents was collected, such as compound name, molecular weight, chemical structure, chromatographic behavior, major mass spectral fragmentation pathway, and the chemical family to which it belongs. It is claimed that natural flavonoids and phenylpropanoids which possessed similar structure of C6–C3 was formed via shikimic acid pathway. Moreover, many phytochemical compounds containing the same carbon skeleton can be structurally divided into groups, and these similar compounds frequently generate the same product ions during the MS/MS process. Secondly, we screened the common fragments from all experimentally generated ions according to the aforementioned summary, which could be defined as structure-unknown diagnostic fragment ions. Thirdly, the database (e.g., Pubchem https:// pubchem.ncbi.nlm.nih.gov/ and Chemspider website http://www. chemspider.com) is employed to search for compound structure based on the predicted chemical formula. Finally, the structure of the compound is tentatively or unambiguously deduced by chromatographic behavior, MS fragment data, biosynthetic pathway, and by comparisons with the authentic reference standards, as well as database-matching (e.g. metlin https://metlin.scripps. edu/landing page.php?pgcontent=mainPage and massbank http:// www.massbank.jp/index.html). The compounds identified in PC are classified into three groups including organic acids and derivatives, flavonoids and phenylpropanolds. Fig. 1 shows the total ion

3.1.3. Identification of flavonoids The flavonoids in PC, containing flavones, flavonols, flavan-3ols, and biflavones, were identified and deduced in accordance with the main aglycone diagnostic ions apigenin, quercetin, keampferol, isorhamnetin, myricetin, catechin and epicatechin as well as the retro-Diels–Alder fragmentation pattern. Compound 16 was identified as epicatechin based on its retention time and [M−H]− ion at

Fig. 1. Total ion chromatogram of PC in negative-ionization mode.

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Table 2 Compounds identified in PC by UPLC-ESI-Q-TOF-MS. No.

RT (min)

Precusor ion m/z [M−H]−

Error (ppm)

Molecular Formular

MS/MS fragmentation

Identification

1 2 3 4a

3.327 3.327 3.502 3.578

191.0552 195.0506 133.0136 173.0451

4.72 2.17 4.83 2.66

C7 H12 O6 C6 H12 O7 C4 H6 O5 C7 H10 O5

Quinic acid Gluconic acid Malic acid Shikimic acid

5a 6

4.212 5.634

191.0191 315.0712

3.26 1.80

C6 H8 O7 C13 H16 O9

173.0442, 127.0391 177.0382, 129.0180 115.0022, 89.0234, 71.0139 137.0239, 93.0351, 111.0421,83,0505 111.0092, 87.0083, 85.0282 153.0176, 109.0274

7

6.209

315.0712

1.80

C13 H16 O9

153.0175, 109.0281

8a 9

7.450 7.942

153.0186 577.1352

4.75 −0.09

C7 H6 O4 C30 H26 O12

10

9.255

577.1343

0.87

C30 H26 O12

11 12 13 14 a 15 a 16 a

10.046 10.088 10.772 12.604 14.159 15.024

337.0927 163.0390 289.0714 577.1317 609.1452 289.0707

0.57 6.50 1.35 5.97 1.49 3.71

C16 H18 O8 C9 H8 O3 C15 H14 O6 C30 H26 O12 C27 H30 O16 C15 H14 O6

17 18

15.776 19.887

337.0930 625.1295

−0.32 2.43

C16 H18 O8 C27 H30 O17

19 a 20

20.142 20.846

163.0396 595.1296

2.85 1.58

C9 H8 O3 C26 H28 O16

21 a 22

21.518 23.227

161.0237 521.2022

4.76 1.22

C9 H6 O3 C26 H34 O11

23 a

23.485

463.0882

−3.45

C21 H20 O12

24 a

24.846

463.0873

0.86

C21 H20 O12

25 26 a 27 28 a

26.500 29.302 29.511 30.677

433.0772 447.0948 433.0764 431.0976

1.40 −3.38 2.87 1.79

C20 H18 O11 C21 H20 O11 C20 H18 O11 C21 H20 O10

109.0286, 91.0171 452.1057, 425.0783, 407.0725, 289.0701 451.0911, 425.0861, 407.0819, 289.0696 191.0545, 173.0457 119.0470, 75.0034 203.0680, 151.0376, 109.0279 407.0830, 289.0663 463,0740, 301,0367 245.0794, 203.0661, 151.0388, 109.0300 191.0545, 173.0457 591.1818, 479.0789, 463.0899, 317.0218 119.0481 463.0842, 300.0257, 271.0258, 178.9936 133.0275, 105.0319 359.1487, 341.1353, 219.0579, 159.0305 317.0276, 287.0171, 178.9962, 151.0014 300.0258, 271.0229, 255.0270, 178.9950, 151.0020 300.0238, 271.0782 301.0262, 271.0230, 255.0289 301.0326 269.0439

29 a

36.150

431.0984

−0.07

C21 H20 O10

285.0381, 255.0287, 227.0323

a

41.698

301.0340

4.56

C15 H10 O7

31 32 33

42.135 46.503 46.517

315.0492 357.1336 269.0452

5.78 0.98 1.28

C16 H12 O7 C20 H22 O6 C15 H10 O5

34 a 35 a 36

47.542 47.604 48.541

285.0396 299.0544 537.0807

3.75 5.70 0.20

C15 H10 O6 C16 H12 O6 C30 H18 O10

37 a

50.269

537.0828

−3.86

C30 H18 O10

38

50.981

537.0822

0.97

C30 H18 O10

39

53.030

739.1665

0.46

C39 H32 O15

285.0383, 255.8217, 178.9789, 151.0022 300.0288, 272.0137 221.0745, 137.0614 241.6543, 221.0287, 117.0335, 107.0119 239.2081, 151.0006 284.0308, 151.0055 443.0412, 417.0593, 375.0485, 331.0556 443.0397, 417.0606, 375.0495, 331.1908, 203.0324 519.0718, 443.0413, 417.0564, 375.0490, 331.0620 593.1192, 447.4982, 300.0235

40

53.897

551.0985

−0.09

C31 H20 O10

41 a

54.367

537.0828

−0.15

C30 H18 O10

42

54.373

551.0989

−0.77

C31 H20 O10

43

58.598

551.0990

−0.93

C31 H20 O10

30

507.1078, 375.0488, 331.0569, 217.0450 493.0920, 469.0849, 376.0526, 284.0342, 253.0447 537.0812, 431.0730, 389.0657, 203.0339, 117.0337 536.0742, 374.0385, 283.0217, 255.0274, 117.0320

Citric acid Protocatechuic acid-hexoside Protocatechuic acid-hexoside Protocatechuic acid B-type procyanidin dimer B-type procyanidin dimer p-Coumaroylquinic acid p-Coumaric acid isomer Catechin Procyanidin B2 Rutin Epicatechin p-Coumaroylquinic acid Myricetin-hexosidepentoside p-Coumaric acid Quercetin-dihexoside Umbelliferone Isolariciresinol-hexoside Myricetrin Isoquercitrin Quercetin-pentoside Quercitrin Quercetin-pentoside Apigenin-7-O-␤-dglucoside Kaempferol-3-ORhamnoside Quercetin Isorhamnetin Matairesinol Apigenin Kaempferol Kaempferide Cupressuflavone Amentoflavone Robustflavone Quercetin-O-di-pcoumaroylrhamnopyranosid Monomethoxylbiflavone Hinokiflavone Monomethoxylbiflavone Monomethoxylbiflavone

The ions in bold values were diagnostic ions. a Structures confirmed by comparison with reference standards.

m/z 289, which matched with the reference compound. Whereas, compound 13 was assigned as catechin, due to its [M−H]− at m/z 289, which generated a fragment ion at m/z 245 owing to

the loss of CO2 residue, and then yielded the fragment m/z 203 ([M−H−CO2 −CH2 CO]− ) owing to cleavage of the ring-structure. Compounds 9, 10 and 14 were characterized as B-type procyani-

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din dimers, due to their [M−H]− ions at 577 m/z and the diagnostic ion at 289 m/z [24]. The fragment ion at m/z 407 represented the elimination of a retro-Diels–Alder fragment and the successive neutral loss of water. Compared with authentic reference standard, compound 14 was definitely identified as procyanidin B2. Quercetin derivatives, compound 15, 20, 24, 25, 26, 27, 30 and 39 were the major flavonols in PC under investigation, according to their common fragment ions at m/z 301 or 300 (deprotonated quercetin), and a fragment of quercetin aglycon at m/z 179. Co-elution with authentic compounds allowed the unequivocal assignment of compounds 15, 24, 26 and 30, as rutin, isoquercitrin, quercitrin and quercetin, respectively. In addition, quercetindihexoside (20) and two quercetin-pentoside (25 and 27) were tentatively identified due to the loss of hexoside (162 Da) and pentoside (146 Da). Compound 39 was also plausibly identified as quercetin-O-di-p-coumaroyl-rhamnopyranosid due to the consecutive loss of 146 Da. Compound 23 was unambiguously identified as myricetrin due to its parent ion [M−H]− at m/z 463 and predominant daughter ion at m/z 317, indicating the loss of rhamnoside in agreement with its MS/MS data of standard compound. Compound 18 was tentatively assigned as myricetin-hexoside-pentoside based on the characteristic fragment ion at m/z 317 (myricetin aglycon), which indicated the neutral loss of 146 and 162 Da. The MS/MS fragmentation demonstrated that the pseudomolecular ions of identified flavonols (compound 29, 34 and 35) were the precursors of kaempferol ([M−H]− at m/z 285 or 284). Compound 34 and 35 were identified as kaempferol and kaempferide based on its retention time and MS/MS spectra, compared with those of corresponding authentic standards. Compound 29, with the parent ion [M−H]− at m/z 431, exhibited a diagnostic daughter ion [M−146]− at m/z 285. It was characterized as kaempferol-3-O-rhamnoside after comparing its retention time and MS data with standard compound. Isorhamnetin (Compound 31) displayed [M−H]− ion at m/z 315 and yielded a predominant fragment at m/z 300 ([M−H−CH3 ]− ) [27]. Compound 28 was identified as apigenin-7-O-ˇ-d-glucoside after comparison of its MS data with that of an authentic standard, while compound 33 was characterized as apigenin for its parent ion [M−H]− at m/z 269 and corresponding fragments [28]. Biflavonoid compound (36–38 and 40–43) in PC could be generally classified into C−C and C−O linked bioflavonoids. The [M−H]− ion of amentoflavone (compound 37) at m/z 537 produced the [M−H−C6 H6 O]− ion at m/z 443, [M−H−C7 H4 O2 ]− at m/z 417, [M−H−C9 H6 O3 ]− at m/z 375 and [M−H−C10 H6 O5 ]− at m/z 331, which was consistent with reference compound. Compound 38, with [M−H]− ion at m/z 537, was characterized as robustflavone. Its MS/MS spectrum showed a fragment at m/z 519 ([M−H−H2 O]− ) corresponding to the intramolecular cyclodehydration, while the other product ions were similar with the amentoflavone-type biflavone [29]. The fragmentations of hinokiflavone (compound 41) indicated a great distinction with the former type of C−C biflavonoids since the two flavone parts are connected via a C−O bond. The product ions at m/z 269 and 253 resulted from the rupture of C−O bond, and the fragment ions at m/z 493 ([M−H−CO2 ]− ) and 469 ([M−H−C3 O2 ]− ) were in agreement with standard solution. Compound 36 was tentatively deduced as cupressuflavone for the [M−H]− ion at m/z 537 according to the reference literature [16]. Meanwhile, compound 40, 42 and 43, possessing key fragment ions at m/z 536 or 537 and secondary fragments, were reasonably characterized as monomethoxylbiflavone. It was deduced that the hydroxy was substituted by a methoxy group. 3.1.4. Identification of phenylpropanolds Phenylpropanolds including coumarin and lignans were firstly detected in PC. Phenylpropanolds were generated through shikimic acid biosynthetic pathway. Their intermediate and final products

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were formed by shikimic acid and aromatic amino acids such as phenylalanine and tyrosine through the step of deamination, hydroxylation and coupling. The presence of shikimic acid in PC was helpful for the identification of its related products. Compound 21 was unequivocally assigned as umbelliferone by comparison with the reference standard. As shown in Fig. S1, umbelliferone yielded a fragment ion at m/z 133, which underwent a successive loss of CO radical. Matairesinol (compound 32) exhibited [M−H]− ion at m/z 357 and produced an important fragment at m/z 221 ([M−H−C8 H9 O2 ]− ) for the loss of six-membered-ring structure in its MS/MS spectrum (Fig. S2). The product ion at m/z 137 ([M−H−C8 H9 O2 −C4 H4 O2 ]− ) was generated through elimination of five-membered-ring structure. Compound 22 was plausibly identified as isolariciresinol-hexoside according to database and MS/MS analysis of the [M−H]− ion at m/z 521, which yielded a fragment at m/z 359 corresponding to the loss of hexoside moiety (162 Da) and a product ion at m/z 341 due to the loss of water. 3.2. Quantitation of nine flavonoids in PC and PCC by UPLC-DAD 3.2.1. Optimization of extraction conditions Considering the higher contents and various biological activities of flavonoids in PC and PCC, nine flavonoids including myricitrin (1), isoquercitrin (2), quercitrin (3), myricetin (4), afzelin (5), quercetin (6), kaempferol (7), amentoflavone (8) and hinokiflavone (9) are selected as key markers for quantitative analysis and quality evaluation. To extract these flavonoids conveniently and sufficiently, vital factors such as extraction solvents, extraction time, solid-toliquid ratio which might influence the extraction efficiency of the analytes were optimized through single-factor tests. Methanol, 75% aqueous methanol, 50% aqueous methanol, ethanol, 75% aqueous ethanol, and 50% aqueous ethanol were examined as extraction solution. The results indicated that 75% ethanol had the most remarkable extraction efficiency for the analytes. Different extraction time (20, 40, 60 and 120 min) was further investigated, and the results showed that the extraction yield was not significantly affected with the change of extraction time. In consideration of energy saving and high efficiency, 60 min was selected in this study. As for the solvent volume, extraction efficiency of total flavonoids appeared an increasing trend with the decrease of solid-liquid ratio. Since the contents of isoquercitrin and afzelin were difficult to accurately quantify when the ratio between plant material weight and solvent volume was 25 mg/mL, solid-liquid ratio of 50 mg/mL was finally selected. Based on these results, the final extraction conditions were determined as follows: 0.5 g sample was extracted by sonication with 75% ethanol of 10 mL for 60 min, which was adequate and appropriate for the analysis. 3.2.2. Optimization of UPLC conditions A segmental monitoring method based on UPLC-variable wavelength detection was performed for simultaneous quantification of nine flavonoids in PC and PCC samples. The UV detector was monitored at 254 nm for flavone glycoside and three flavonoid aglycones (myricitrin (1), isoquercitrin (2), quercitrin (3), myricetin (4), afzelin (5), quercetin (6), kaempferol (7) at 0–14 min and 340 nm for biflavones (amentoflavone (8) and hinokiflavone (9)). The chemical structures of analytes are showed in Fig. 2. In order to achieve a good separation of the analytes, various flow rate (0.7, 0.8 and 0.9 mL/min) and column temperature (25, 35 and 40 ◦ C) were systematically compared. Moreover, formic acid (0.1% and 0.2%, v/v) was added into the mobile phase to improve the peak shape and restrain the peak tailing. Consequently, flow rate of 0.8 mL/min, column temperature of 35 ◦ C, the detection wavelength of 254 nm (0–14 min) and 340 nm (14–24 min) and mobile phase system (0.2% aqueous formic acid (A) and acetonitrile (B)) provided good sepa-

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Fig. 2. The chemical structures of nine flavonoids assayed.

ration and strong UV absorption for most target compounds in PC sample. The typical LC chromatograms are shown in Fig. 3.

3.2.3. Method validation The developed UPLC-DAD method was validated by determination of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, repeatability, stability and recovery. For the calibration curves, seven or eight working solution concentrations were analyzed. As shown in Table S1, all standard curves for nine

compounds showed good linearity with correlation coefficients (r2 ) higher than 0.9995 in a relatively wide concentration range. The limits of detection (LODs) and quantification (LOQs) were 0.15-0.56 ␮g/mL and 0.30-1.55 ␮g/mL, which were determined by injecting serial diluted standard working solutions and taking generated peaks with signal-to-noise ratio of 3 and 10 as criteria, respectively. The precision of the method was evaluated by the determination of intra- and inter-day variances. The intra-day variability test was analyzed at three different concentration levels

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Fig. 3. HPLC chromatograms of nine flavonoids, (A) reference compounds, (B) PC sample and (C) PCC sample (1, myricitrin; 2, isoquercitrin; 3, quercitrin; 4, myricetin; 5, afzelin; 6, quercetin; 7, kaempferol; 8, amentoflavone; 9, hinokiflavone).

(low, medium, high) with three replicates at each level on the same day, whereas the inter-day variability test was carried out in triplicates on the consecutive three days. As shown in Table S2, RSDs ranged from 0.03 to 4.18%. For the repeatability test, six replicates of the same batch samples were extracted and analyzed. To confirm the stability, the same sample was stored at room temperature and evaluated by replicate injection at 0, 2, 4, 8, 12 and 24 h, the result (Table S3) showed the sample solution is stable within 24 h. Recovery was used to further evaluate the accuracy of the method by calculating the mean recoveries of the analytes. The overall recoveries of all the compounds were ranged from 96.52% to 104.03% with RSD values less than 4.09%. The verified analytical method was sen-

sitive, repeatable, and accurate for the simultaneous determination of the nine flavonoids.

3.2.4. Samples analysis A total of 39 batches of PC samples from different origins, along with 10 batches of PCC were collected, prepared and analyzed by the method described above. The quantitative results are presented in Table S4. It was noticeable that the content of each constituent differs greatly among the raw and processed PC samples. Among the nine flavonoids assayed, quercitrin was the most abundant compound and its content fell in the range of 1.03–4.71 mg/g in PC, which is accordance with the standard of the Chinese Phar-

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Fig. 4. The total contents of nine analytes in 49 batches of samples (Each color represents a different compound).

higher than that in PCC, as their contents ranged from 0.20 mg/g to 0.27 mg/g and 0.07 mg/g to 0.19 mg/g, respectively. Compared with the literature, the contents of compounds in PCC in our study exhibited higher batch-to-batch similarity since our crude materials were carbonized under the same condition. Moreover, quercetin was the primary component that only appeared in the charcoal of PC in all studies [30]. It was deduced that such chemical discrimination could be attributed to thermal decomposition during the process of high-temperature treatment. Certain flavonoid glycosides would break down, resulting in the increase of aglycones compounds in PCC products. It was also found that the flavonoid contents in PC3, PC11 as well as PC27 were much lower than other PC samples. To visualize the classification trends in PC samples, HCA was conducted based on the contents of nine bioactive flavonoids, which could divide tested samples into different categories. As shown in Fig. 5, these samples were sorted into two clusters, representing the diversity in quality of PC samples. PC3, PC11 and PC27, which were collected from Zhejiang, Shandong and Guangxi in turn could be included in Group 2, while the other batches were grouped into the same class (Group1). It’s evident that PC11 collected from Shandong was clearly different from the other batches PC8-10, 12 and 13 in Group 1, which were also from Shandong. The results illustrated that there is no significant resemblance among PC samples from the same geographical origins. It was concluded that sample quality classification might be mostly relevant to harvest time and geographical climate and processing method rather than origins. Standardization of planting, harvesting, and processing should be intensively proposed to assure the quality consistency of PC. 4. Conclusion In this work, the data on qualitative phytochemical composition of PC by UPLC-Q-TOF-MS/MS indicated the presence of a huge variety of flavonoids and organic acids. Coumarin and lignan were firstly identified from PC. The quantitative analysis of nine flavonoids by UPLC-DAD and HCA demonstrated that the content of each constituent differs greatly among the raw and processed PC samples. This is the first report on simultaneous qualitative and quantitative analysis of chemical constituents in PC. The results obtained from this study would be helpful in establishing a scientific and rational quality control method for PC. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81673569, 81473343), the Scientific Research Foundation of Hebei Province Education Department (No. QN2017093) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data

Fig. 5. Dendrograms of hierarchical cluster analysis for 39 PC samples based on the content of nine flavonoids.

macopoeia (2015 edition, >0.10% w/w). In contrast, the contents of isoquercitrin and afzelin in PC were rather lower. The results in Fig. 4 also indicated that contents in PC (the mean value was 7.40 mg/g) were significantly higher than those in PCC (the mean value was 1.21 mg/g). Flavone glycoside myricitrin, isoquercitrin, quercitrin, myricetin and afzelin were only detected in PC, whereas flavonoid aglycones myricetin, quercetin and kaempferol were merely quantified in PCC samples. The contents of amentoflavone (0.56–1.07 mg/g) and hinokiflavone (0.62–1.03 mg/g) in PC were

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